PROJECT SUMMARY/ABSTRACT Multiple sclerosis (MS) and other autoimmune diseases constitute a major healthcare burden at a cost of >$50 billion per year. Autoimmunity arises from a failure of immunoregulation, in which regulatory T cells (Tregs) play a crucial role in balancing immune responses, though their suppressive mechanisms are incompletely understood and little is known about their cellular dynamics. Our overall goal is to identify cellular and molecular immunoregulatory mechanisms that contribute to disease progression and response to therapy. Building on our expertise in two-photon (2-P) imaging at the cellular level, and Ca2+ signaling at the molecular level, we will use the experimental autoimmune encephalomyelitis (EAE) mouse model of MS to investigate cellular interactions and molecular mechanisms underlying disease progression, as well as therapeutic approaches to promote remission. We focus in particular on Tregs, which maintain homeostasis and limit autoimmunity. Our central hypothesis is that Tregs limit autoimmune-mediated demyelination in the EAE model at two levels. (i) At the cellular level, Tregs compete with conventional T cells for access to antigen-presenting dendritic cells (DCs), and restrict egress of differentiated, pathogenic effector T cells (Teffs) from lymph nodes (LN). In Aim 1, we will apply simultaneous 2-P imaging of Tregs, nave T cells, Teffs, and DCs in the LN to reveal fundamental cell trafficking and interaction dynamics during EAE induction, progression, and remission. Aim 2 extends those studies to the spinal cord where, by additionally imaging oligodendrocytes and neuronal cells, we will elucidate the cellular dynamics of neuroinflammation and demyelination during disease progression and remission. In both Aims, we further propose to define cellular dynamics during therapies that show great promise for treating MS in humans including drugs that target S1P1 receptors to cause lymphocyte sequestration within the LN, and stem cell therapy to promote remyelination. (ii) At the molecular level, Tregs directly contact target lymphocytes to inhibit Ca2+ signaling and suppress their activation. We have previously shown that Ca2+ signaling in T cells is mediated by plasma membrane Orai1 channels and triggered by STIM1 in the endoplasmic reticulum. In Aim 3, we propose to investigate the roles of these proteins employing a novel `toolkit' of genetically-encoded Ca2+ indicators and probes of channel function to monitor cellular Ca2+ signaling in LN and spinal cord. We hypothesize that Treg contact with nave or effector T cells results in dissolution of Orai1 puncta and transendocytosis of Orai1 channel protein into Tregs. We will evaluate Orai1 as a therapeutic target in MS by visualizing cellular dynamics following administration of a specific Orai1 blocker during EAE and the translational potential of this approach will be validated with human cells. Although this proposal is targeted specifically to MS, our findings and novel immunoimaging approaches will contribute in a broader context to a better understanding of how immune responses are initiated, how immunological tolerance is achieved, how Tregs prevent autoimmunity and dampen immune responses, and how autoimmunity and infectious diseases can be effectively treated.